Compositions and methods for reducing at least one symptom of human allergy to cats

ABSTRACT

Compositions and methods reduce symptoms of human allergy to cats. The effectiveness of a molecule which specifically binds to Feline domesticus allergen number 1 (Fel D1) is enhanced by prolonging the time the immunoglobulin stays within the mouth of a cat to whom the anti-Fel D1 molecule is administered in a pet food. The compositions and methods use a powder that is a dried hydrogel encapsulating the anti-Fel D1 molecule, and the hydrogel is based on gelatin, collagen peptides, or gelatin and collagen peptides; and carrageenan. The methods of making the powder provide a high encapsulation efficacy, sustained release of the anti-Fel D1 molecule in the cat&#39;s mouth, and oral adhesion of the anti-Fel D1 molecule in the cat&#39;s mouth.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/874,102, filed Jan. 18, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/449,883 filed Jan. 24, 2017, eachof which is incorporated by reference herein in its entirety.

BACKGROUND

Approximately 20% of adults suffer from allergy to cats and/or theirdander. Symptoms of cat allergies range from mild rhinitis andconjunctivitis to life-threatening asthmatic responses, and catallergies are a major roadblock to cat ownership. For example, catallergy is the primary reason given by cat owners for returning cats toanimal shelters.

Most cat allergies are caused by a small stable glycoprotein called FelD1 (Feline domesticus allergen number 1). This protein is transferred tocat dander by their grooming process and becomes airborne. Uponinhalation of cat dander having Fel D1 attached, an allergy cascade istriggered because of the recognition of the Fel D1 by human immunecells.

SUMMARY

The present disclosure is directed to oral adhesion/mucoadhesion of amolecule which specifically binds to Fel D1, hydrogel technology forcontrolled release of the anti-Fel D1 molecule, and processingapproaches to create a powder from the hydrogel. This anti-Fel D1molecule (e.g., an egg immunoglobulin such as IgY from an avianimmunized with Fel D1) can reduce at least one symptom of human allergyto cats because it blocks the recognition of the Fel D1 by the humanimmune cells. The proposed site of action of this anti-Fel D1 moleculeis within the cat's mouth where the Fel D1 protein is secreted and/or onthe cat's fur where the Fel D1 is deposited during the grooming process.

The present inventors propose delivery of the anti-Fel D1 molecule tocats via their food. The initial animal trials indicated thatadministration of this molecule to cats via their food only results inan approximately 30-35% reduction in free Fel D1 in cat saliva. Thepresent inventors believe that the main limitation is the need for theanti-Fel D1 molecule to stay in the cat's mouth for a long period oftime, but the process of eating and drinking results in a large amountof the molecule administered in food being swallowed and thus not havingan effect. Increasing IgY oral residence can be measured in two distinctways. In one aspect, the IgY can be measured as having a higherconcentration at a specific point in time, e.g., after 5 minutes ofeating. In another aspect, the IgY can be measured as taking longer todecrease below a certain threshold, e.g, more than 10 ug/ml, ≥1 ug/ml oreven >100 ng/ml.

The present inventors believe that one of the primary challenges increating a controlled-release hydrogel for delivery in a cat's mouth isto ensure that the hydrogel dissolve instantaneously, not melt, withinthe cat's mouth, has mucoadhesive properties, and is appealing to cats.Chitosan is a well-known highly mucoadhesive polymer but is mucoadhesiveat acidic pHs, and cats' mouths and typical pet foods have neutral pHs.Another leading mucoadhesive polymer is porcine gelatin, but gelscreated from gelatin melt at body temperatures.

As detailed in the experimental results disclosed later herein, thepresent inventors surprisingly found that an additional biopolymer cancontrol the melting/dissolution of a gelatin-based hydrogel.Specifically, wet hydrogels that were gelatin/kappa-carrageenan co-gelsprovided slower release of an anti-Fel D1 immunoglobulin (IgY). Thisfinding was unexpected because combinations of biopolymers do nottypically lead to good gels because the two polymers undergo phaseseparation. Lundin, L. O., Odic, K. and Foster, T. J. (1999) Phaseseparation in mixed carrageenan systems. Proceedings to Supermolecularand Colloidal Structures in Biomaterials and Biosubstrates, Mysore,India.

Accordingly, in one embodiment, a food grade powder providing sustainedrelease of an anti-Fel D1 molecule is provided. The food grade powdercan comprise a dried hydrogel and the anti-Fel D1 molecule; wherein thedried hydrogel comprises gelatin, collagen peptides, or gelatin andcollagen peptides; and carrageenan; wherein the anti-Fel D1 molecule isencapsulated in the dried hydrogel.

Additionally, in an embodiment, the present disclosure provides a methodof making a food grade powder providing sustained release of an anti-FelD1 molecule. The method comprises: heating a solution comprisinggelatin, carrageenan and the anti-Fel D1 molecule; forming droplets ofthe heated solution; gelling the droplets by subjecting the droplets toa gelling bath comprising salt or oil to form gelled beads in which theanti-Fel D1 molecule is encapsulated; and drying the gelled beads toform the food grade powder.

The droplets can be formed by passing the heated solution through anozzle. The heating of the solution can comprise subjecting the solutionto a temperature of about 60° C. The gelling bath can comprise about 100mM of potassium chloride. The drying can comprise subjecting the beadsto fluid bed drying.

The present inventors also noted that the most common approach to dryingin the food industry is spray drying but found that conventional spraydrying of gelatin or gelatin/carrageenan compositions lead to rapidrelease of an anti-Fel D1 molecule (IgY) during in vitro testing due toa highly porous powder structure. Surprisingly, the present inventorsfound that when the compositions were gelled before drying, release ofthe anti-Fel D1 molecule during in vitro testing was considerablyslower. For example, an extrusion process led to a higher percentage ofencapsulation and a greater ability to control the final size of thepowder particle. Interestingly, the extrusion process resulted in a muchbetter structure (smoother surface, fewer inclusions) when carrageenanwas incorporated into the mixture prior to extrusion.

Accordingly, in an embodiment, the present disclosure provides a methodof making a food grade powder providing sustained release of an anti-FelD1 molecule. The method comprises: heating a solution comprisinggelatin, carrageenan, and the anti-Fel D1 molecule; extruding the heatedsolution to cool the heated solution and form an extruded hydrogel inwhich the anti-Fel D1 molecule is encapsulated; and drying the extrudedhydrogel.

The method can comprise milling the dried hydrogel to a predeterminedsize. The heating of the solution can comprise subjecting the solutionto a temperature of at least about 60° C. before the extruding and canfurther comprise subjecting the solution to a holding tank having atemperature of at least about 40° C. and then transferring the solutionfrom the holding tank to another device which subjects the solution tothe temperature of at least about 60° C. The extruding can be performedby a tubular heat exchanger having a temperature of about 5° C. to about55° C., or in one aspect, of about 5° C. to about 35° C., and at least aportion of the heating of the solution can be conducted in a funneland/or a pump of the tubular heat exchanger. The method can compriseimmersing the extruded hydrogel in a composition comprising calciumbefore the drying. The drying can comprise subjecting the extrudedhydrogel to a drying tunnel, oven, or fluid bed drier.

In another embodiment, the present disclosure provides a food gradepowder providing sustained release of an anti-Fel D1 molecule. The foodgrade powder comprises a dried hydrogel comprising gelatin, collagen, orgelatin and collagen peptides; carrageenan; and the anti-Fel D1molecule; where the anti-Fel D1 molecule is encapsulated in the driedhydrogel. The powder can be made by one of the two methods noted above.

The present inventors also noted that it is well known that spray dryinggelatin is very difficult. The reason is that gelatin solutions at hightotal solids have high elasticity which forms long filaments during theatomization of spray drying. The formation of filaments during the spraydrying process results in a powder which has very low porosity and isvery fluffy, and both of these features severely limit powderflowability and processing capabilities.

The present inventors surprisingly found that optimized spray dryingconditions and also careful selection of gelatin properties (e.g.,molecular weight) and wall materials could develop a formulation capableof being spray dried, with the resulting powder retaining itsmucoadhesive properties. Notably, a powder which is created simply bydry mixing the gelatin and the anti-Fel D1 molecule will not result inincreased oral retention of the anti-Fel D1 molecule because theanti-Fel D1 molecule is not encapsulated.

Accordingly, in an embodiment, the present disclosure provides a methodof making a food grade powder providing sustained release of an anti-FelD1 molecule. The method comprises spray drying a solution comprising ananti-Fel D1 molecule and 5.0 to 20.0 wt. % of gelatin having a bloomvalue of 40 to 300, in one aspect, 40 to 300, or even about 100, to forma dried hydrogel in which the anti-Fel D1 molecule is encapsulated.

The resultant powder can be a food grade powder providing sustainedrelease of an anti-Fel D1 molecule and comprising a dried hydrogelcomprising the anti-Fel D1 molecule and 0.5 to 90 wt. % of gelatin. Theanti-Fel D1 molecule is encapsulated in the dried hydrogel, and thepowder can have a density of about 0.265 to about 0.3 g/cm³. In anembodiment, the gelatin can be Type A porcine gelatin having a positivecharge at a pH below 7.0 and can be the only biopolymer capable ofgelling below 60° C. in the dried hydrogel. In one embodiment, thegelatin and collagen peptides can have a molecular weight between 2,000and 1 million Daltons and/or a bloom value of 40 to 300.

The present disclosure also provides a method of reducing symptoms ofhuman allergy to a cat. The method comprises orally administering to thecat an effective amount of any of the embodiments of the food gradepowder disclosed herein and/or a food grade powder resulting from any ofthe methods of making a food grade powder disclosed herein. The powdercan be administered as part of a pet food further comprising at leastone component selected from the group consisting of protein, fat,carbohydrate, vitamin and mineral.

An advantage of one or more embodiments provided by the presentdisclosure is to reduce, minimize or prevent at least one symptom of anallergic reaction to a cat in a sensitized human.

Another advantage of one or more embodiments provided by the presentdisclosure is to reduce, minimize, or prevent allergies caused by cats.

Another advantage of one or more embodiments provided by the presentdisclosure is to expose a cat's mouth to a molecule that binds FelD1before it contacts a sensitive human; a bound allergen cannot interactwith the mast cells in the human and thereby cannot cause an allergenicreaction.

A further advantage of one or more embodiments provided by the presentdisclosure is a carrier/delivery system that enhances the oral residencetime of an anti-Fel D1 molecule in a cat's mouth and is highly appealingto the cat (e.g., administrable in a pet food).

A further advantage of one or more embodiments provided by the presentdisclosure is to increase owner appeal, increase cat ownership, andimprove the health of adults and children in cat-owning households.

Still another advantage of one or more embodiments provided by thepresent disclosure is to use multiple technologies to reduce symptoms ofhuman allergy to cats.

Yet another advantage of one or more embodiments provided by the presentdisclosure is to control the melting/dissolution of a hydrogelencapsulating the active molecule.

Another advantage of one or more embodiments provided by the presentdisclosure is to address cat allergy using a diet of the cat and therebyreduce or remove the need for the allergic human to take medication oravoid contact with the cat.

A further advantage of one or more embodiments provided by the presentdisclosure is to provide a powder having a high content of an anti-FelD1 molecule due to high encapsulation efficacy (e.g., by using in situgelation instead of gelation bath) and/or minimal loss of the anti-FelD1 molecule (e.g., by coping with higher viscosity matrices associatedwith lower temperatures).

Still another advantage of one or more embodiments provided by thepresent disclosure is easy formation of extruded encapsulated materialsdue to gelation temperature profile, strand strength, and lubricationproperties.

Additional features and advantages are described herein and will beapparent from the following Detailed Description and the Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic diagram of a system that can be used in anembodiment of a first method of making a food grade powder that reducesat least one symptom of human allergy to cats (the “dripping method”).

FIG. 1B is a schematic diagram of an embodiment of a food grade powdermade by the dripping method.

FIG. 2A is a schematic diagram of a system that can be used in anembodiment of a second method of making a food grade powder that reducesat least one symptom of human allergy to cats (the “extrusion method”).

FIG. 2B contains comparative photographs between an embodiment of a foodgrade powder made by the extrusion method and a food grade powder madeby conventional spray drying.

FIG. 2C is a photograph of an embodiment of a system that can be used inthe extrusion method.

FIG. 2D is a photograph of the system of FIG. 2C in use.

FIG. 3 is a schematic diagram of a system that can be used in anembodiment of a third method of making a food grade powder that reducesat least one symptom of human allergy to cats (the “controlled spraydrying method”).

FIG. 4 is a graph of relative anti-Fel D1 IgY release over time fromhydrogels of different compositions.

FIGS. 5A-5D contain photographs showing the visual appearance ofdifferent hydrogels prepared from solutions of 10 wt. % gelatin type Aand 2 wt. % egg yolk extract. Samples were prepared as: (FIG. 5A) gelpiece 3×20×20 mm, (FIG. 5B) spray dried powder (Diameter, D4,3 32 μm),(FIG. 5C) wet beadlet produced by dripping into oil, and (FIG. 5D) driedbeadlet produced from (III).

FIGS. 6A and 6B contain scanning electron microscopy (SEM) images of thecross section of powders made from solutions of 10 wt. % gelatin and 2wt. % egg yolk extract. The powders were made: (FIG. 6A) by spray dryingor (FIG. 6B) by dripping followed by fluidized bed drying.

FIGS. 7A-D contain SEM analysis of the microstructural complexity of wet(A-C) and dry (D) beadlets. Composition: 9.81 wt. % gelatin (type A),1.99 wt. % active ingredient, 1.47 wt. % WR78 carrageenan, and no salt.(FIG. 7A) freeze fracture cryo-SEM, (FIG. 7B) cryo-SEM with icesublimation, (FIG. 7C) cryo-SEM with extended ice sublimation and (FIG.7D) SEM of dried beadlet.

FIG. 8 is a graph of the impact of different gel preparation methods onanti-Fel IgY release from 10 wt. % gelatin type A hydrogels preparedwith 2 wt. % egg yolk extract. Samples were prepared as: (I) gel piece3×20×20 mm, (II) spray dried powder (Diameter, D4,3 32 μm), (III) wetbeadlet produced by dripping into oil, and (IV) dried beadlet producedfrom (III).

FIGS. 9A and 9B contain scanning electron micrographs of powders (A) and(B) produced by spray drying solutions of 10 wt. % gelatin, 1 wt. %kappa-carrageenan and 2 wt. % egg yolk extract.

FIGS. 10A-10C contains photographs (A and B) and a scanning electronmicrograph (C) of wet and dry beadlets produced by spray dryingsolutions of 10 wt. % gelatin (type A 280 bloom), 1 wt. %kappa-carrageenan and 2 wt. % egg yolk extract.

FIGS. 11A and 11B contain graphs showing the impact of different gelpreparation methods on IgY release from 10 wt. % gelatin/1 wt. %kappa-carrageenan co-hydrogels prepared with 2 wt. % egg yolk extract.Samples were prepared as: (I) wet beadlets produced by dripping into 100mM KCl solution, (II) dried beadlets produced from (I), (III) wetbeadlets produced by dripping 10 wt. % gelatin (type A) and 2 wt. % eggyolk extract into oil, and (IV) dried beadlets produced from (III).

FIG. 12 contains graphs of experimental data regarding denaturation andencapsulation efficiency of anti-Fel D1 IgY encapsulated by the drippingand extrusion methods.

FIGS. 13A and 13B contain graphs of experimental data regardingcontrolled release of anti-Fel D1 IgY encapsulated by the dripping andextrusion methods.

FIG. 14 is a graph of experimental data regarding oral adhesion ofanti-Fel D1 IgY encapsulated by the extrusion method.

FIG. 15 is a graph of experimental data regarding oral residence time invivo of unencapsulated anti-Fel D1 IgY.

FIG. 16 is a graph of experimental data regarding oral adhesion ofanti-Fel D1 IgY encapsulated by the controlled spray drying method.

FIG. 17 is a table showing the composition and powder properties(physical appearance and density) of different spray dried gelatinpowders. Powder quality index: 1—can't pump liquid, 2—blocked nozzle,3—extensive spider webs, 4—fluffy powder, 5—medium dense power, 6—densepowder.

FIG. 18 is a schematic showing the design of the clinical trial used toassess the impact of encapsulation on IgY oral retention.

FIG. 19 is a table showing the composition of the two test samples usedin the human clinical trial used to assess the impact of encapsulationon IgY oral retention.

FIG. 20 is a graph showing the change in human saliva IgY concentrationverses time across the two treatments i) 140 mg IgY delivered in 1 g eggpowder, ii) 140 mg of IgY delivered as a spray dried powder of gelatin(type A, 100 bloom) and defatted egg yolk proteins. Error bars represent95% confidence intervals n=12 participants.

DETAILED DESCRIPTION Definitions

As used in this disclosure and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. The words “comprise,” “comprises” and “comprising”are to be interpreted inclusively rather than exclusively. Likewise, theterms “include,” “including” and “or” should all be construed to beinclusive, unless such a construction is clearly prohibited from thecontext. However, the devices disclosed herein may lack any element thatis not specifically disclosed. Thus, a disclosure of an embodiment usingthe term “comprising” includes a disclosure of embodiments “consistingessentially of” and “consisting of” the components identified.

The term “and/or” used in the context of “X and/or Y” should beinterpreted as “X,” or “Y,” or “X and Y.” Where used herein, the terms“example” and “such as,” particularly when followed by a listing ofterms, are merely exemplary and illustrative and should not be deemed tobe exclusive or comprehensive. Any embodiment disclosed herein can becombined with any other embodiment disclosed herein unless explicitlystated otherwise.

Ranges are used herein in shorthand to avoid listing every value withinthe range. Any appropriate value within the range can be selected as theupper value or lower value of the range. Moreover, the numerical rangesherein include all integers, whole or fractions, within the range.

All percentages expressed herein are by weight of the total weight ofthe composition unless expressed otherwise. When reference is made tothe pH, values correspond to pH measured at 25° C. with standardequipment. As used herein, “about” or “substantially” in reference to anumber is understood to refer to numbers in a range of numerals, forexample the range of −10% to +10%, −5% to +5%, −1% to +1%, or in oneaspect, −0.1% to +0.1% of the referenced number.

“Food grade” means that the powder is edible by a cat and is not toxicto the cat. For example, a food grade powder contains a maximum of 5.0wt. % of glycerol.

The term “allergy” is synonymous with “allergic response” or “allergicreaction.” Each of the terms refers to a state of immune responsivenessin an animal specific to an exogenous antigen (or “allergen”) that isnot otherwise harmful to the animal. A “symptom” of an allergic responserefers to any measure of the immune responsiveness, e.g., on themolecular level (including measurement of an activity or expression of aprotein, or transcript or gene), the cellular level, the organ level,the systemic level, or the organism level. Such symptoms can compriseone or more such levels. “Reducing at least one symptom” includesreducing such symptoms before they occur so that there are no symptomsto an allergic response and thus preventing the allergic response.

Symptoms may include generalized phenomena such as inflammation,respiratory complaints, swelling, or distress typically associated withallergy, rhinitis, edema, and allergic skin disorders including but notlimited to atopic dermatitis (e.g., eczema), urticaria (e.g., hives) andangioedema, and allergic contact dermatitis. More specific phenomenathat are “symptoms” of an allergic response include any measurable orobservable change, for example at the cellular level, including but notlimited to local or systemic changes in cell populations, eosinophilia,recruitment and/or activation of immune cells, including, for example,mast cells and/or basophils, changes in antigen-presenting cells(including but not limited to FcϵRI-bearing dendritic cells),intracellular or molecular changes, including measurement orobservations of one or more steps in an immunological cascade, releaseof intracellular compounds that mediate an allergic response (e.g.,mediators), and changes in one or more cytokines (e.g., IL-3, IL-5,IL-9, IL-4, or IL-13) or related compounds or antagonists thereof. Theskilled artisan will understand that certain symptoms as defined hereinas more readily measured than others, and some are measured throughsubjective assessment or self-assessment of the symptom. For othersymptoms, there are convenient or rapid assays or measurements forobjectively assessing changes.

As used herein, an “effective amount” is an amount of any thecompositions disclosed herein administered to a cat that reduces atleast one symptom of cat allergy in a sensitized human in the sameenvironment as the cat (e.g., a house, room, car, office, hotel, yard,garage). The relative term “reducing at least one symptom” and similarterms refer to a reduced severity resulting from the compositions andmethods disclosed herein relative to the severity if the compositionsand methods are not used but conditions are otherwise identical. As usedherein, “reducing at least one symptom” includes, but is not limited to,reducing such symptoms before they occur so that there are no symptomsto an allergic response and thus preventing the allergic response.

As used herein, an “anti-Fel D1 molecule” is any molecule able tospecifically bind Feline domesticus allergen number 1 (Fel D1), forexample an antibody, an aptamer, an agonist/antagonist of Fel D1, orportions of such molecules (e.g., an antigen binding fragment (Fab) ofan antibody). The term “antibody” includes polyclonal and monoclonalantibodies of any type and from any species, as well as immunoglobulinfragments such as Fv, Fab, Fab′, F(ab′)₂, or other antigen-bindingantibody fragments, sequences or subsequences that interact withmolecular specificity (e.g., demonstrate specific binding) with anantigen.

In one embodiment, the anti-Fel D1 molecule is an antibody (e.g., IgY)produced by immunizing an avian such as a chicken with Fel D1 to causeproduction of the antibody in eggs. The antibodies can be separated fromthe egg and administered to the animal; or the eggs and/or a part of theeggs such as the egg yolk can be applied directly onto or admixed with afood or other composition suitable for administration to an animal. Inone aspect, the anti-Fel D1 molecule can be one of the embodiments ofthe molecules disclosed in U.S. Pat. No. 8,454,953 to Wells et al.,“Methods for reducing allergies caused by environmental allergens,”incorporated herein by reference in its entirety.

The methods and devices and other advances disclosed herein are notlimited to particular methodologies, protocols, and reagents because, asthe skilled artisan will appreciate, they may vary. Further, theterminology used herein is for the purpose of describing particularembodiments only and does not limit the scope of that which is disclosedor claimed.

Unless defined otherwise, all technical and scientific terms, terms ofart, and acronyms used herein have the meanings commonly understood byone of ordinary skill in the art in the field(s) of the presentdisclosure or in the field(s) where the term is used. Although anycompositions, methods, articles of manufacture, or other means ormaterials similar or equivalent to those described herein can be used,specific devices, methods, articles of manufacture, or other means ormaterials are described herein.

EMBODIMENTS

The present disclosure relates generally to compositions and methods ofusing an active molecule that reduces at least one symptom of humanallergy to cats. More specifically, the present disclosure is directedto enhancing the effectiveness of the active molecule by prolonging thetime the active molecule stays within the mouth of a cat administeredthe active molecule and/or increasing the concentration of activemolecule detected in the mouth. Increasing the active molecule, e.g.,IgY, oral residence can be measured in two distinct ways. In one aspect,the active molecule can be measured as having a higher concentration ata specific point in time, e.g., after 5 minutes of eating. In anotheraspect, the active molecule can be measured as taking longer to decreasebelow a certain threshold in the oral cavity, e.g., more than 10 ug/ml,≥1 ug/ml, or even >100 ng/ml.

An aspect of the present disclosure is a food grade powder that providessustained release of an anti-Fel D1 molecule. The anti-Fel D1 moleculeis encapsulated in the dried hydrogel.

In an embodiment, the food grade powder comprises a dried hydrogelcomprising gelatin, carrageenan and the anti-Fel D1 molecule. In oneaspect, the gelatin and the carrageenan are the only biopolymers capableof gelling below 60° C. in the dried hydrogel. In another aspect, thegelatin and the carrageenan are the only non-allergy reducingbiopolymers. The gelatin can be from any source, such as pig, beef, orfish. In one aspect, the gelatin used with the carrageenan is Type Agelatin (acid-treated), has a positive charge and has a moderatemolecular weight between 2,000 and 2,000,000 Daltons (i.e. 40 to 300bloom), or in one aspect between 100 to 200 bloom. For example, in onespecific embodiment is porcine gelatin having a positive charge at pHsbelow 7.0.

The carrageenan can be any carrageenan from an algal or vegetable source(e.g., kappa, iota or gamma carrageenan). In one aspect, the carrageenangels can include salt ions (e.g. potassium, calcium, magnesium, sodium).For example, the carrageenan can be kappa carrageenan, iota carrageenan,a co-polymer of kappa carrageenan and iota carrageenan or a mixture ofkappa carrageenan and iota carrageenan. One aspect is a carrageenan thatgels in the presence of potassium, i.e., kappa carrageenan or aco-polymer of kappa carrageenan and iota carrageenan.

The food grade powder can comprise a source of the anti-Fel D1 molecule.In one aspect, the anti-Fel D1 molecule can be anti-Fel D1 IgY, and asource can be partially defatted egg yolk powder from an avian immunizedwith Fel D1. The powder can comprise 0.5 to 90 wt. % of the gelatin, 0.1to 50 wt. % of the carrageenan and 10 to 99.5 wt. % of the source of theanti-Fel D1 molecule; in one aspect, 10.0 to 80.0 wt. % of the gelatin,0.5 to 20 wt. % of the carrageenan and 20 to 89.5 wt. % of the source ofthe anti-Fel D1 molecule; and in one specific aspect, 20.0 to 80.0 wt. %of the gelatin, 1 to 10 wt. % of the carrageenan and 19 to 79 wt. % ofthe a source of the anti-Fel D1 molecule; and in still another aspect,25.0 to 60.0 wt. % of the gelatin, 1 to 10 wt. % of the carrageenan and39 to 74 wt. % of the a source of the anti-Fel D1 molecule.

Another aspect of the present disclosure is a method of making a foodgrade powder providing sustained release of an anti-Fel D1 molecule, forexample the powder comprising gelatin, carrageenan and an anti-Fel D1molecule as disclosed above. The method can comprise heating a solutioncomprising gelatin, carrageenan and the anti-Fel D1 molecule, in oneaspect, to a temperature of about 60° C. The method can further compriseforming droplets of the heated solution, for example by passing theheated solution through a nozzle. The matrix material (the gelatin andthe carrageenan) can be atomized by the nozzle. FIG. 1A is a schematicdiagram of a non-limiting example of a suitable apparatus for performingthis method, and FIG. 1B is an illustration of the active compound (theanti-Fel D1 molecule) encapsulated in the gelatin-carrageenan co-polymerformed by this method.

The droplets can be gelled by subjecting the droplets to a gelling bathcomprising salt or oil, for example by dripping, jetting or prilling thedroplets into the bath, to form gelled beads in which the anti-Fel D1molecule is encapsulated. In an embodiment, a syringe pump or volumetricpump pushes the heated solution through the nozzle into the gellingbath.

The bath can comprise about 25-100 mM of potassium chloride or 100 mMcalcium chloride, or both. In this regard, the present inventors foundthat some embodiments in which potassium chloride concentrations higherthan 100 mM were used lead to carrageenan melting points which are toohigh and thereby increase processing temperatures and cause considerableloss (30-80%) of activity of the anti-Fel D1 molecule due todenaturation. In some embodiments, the potassium chloride can bepartially or completely replaced by calcium chloride.

The gelled beads can be collected, optionally washed, and dried to formthe food grade powder. In one aspect, the gelled beads can be dried byfluid bed drying. For example, the washed wet beads can be dried in afluid bed drier with an inlet air temperature of 25° C. at an air flowof 80 m³/minute for 3 to 5 hours.

Although the method detailed above is suitable for making a food gradepowder providing sustained release of an anti-Fel D1 molecule, thepresent inventors discovered an extrusion process in which a higherpercentage of the anti-Fel D1 molecule is encapsulated and there is agreater ability to control the final size of the powder particle.Furthermore, this extrusion process surprisingly resulted in much betterstructure (e.g., smoother surface, fewer inclusions) when carrageenanwas incorporated into the mixture prior to extrusion.

Therefore, the present disclosure provides another embodiment of amethod of making a food grade powder providing sustained release of ananti-Fel D1 molecule and comprising gelatin, carrageenan and theanti-Fel D1 molecule. The method can comprise heating a solutioncomprising gelatin, carrageenan, and the anti-Fel D1 molecule in aholding tank that has a temperature of at least about 40° C. Then thesolution can be heated to a temperature of at least about 50-60° C., forexample in a funnel and/or a pump of a tubular heat exchanger. Then thetubular heat exchanger can cool the heated solution and extrude ahydrogel in which the anti-Fel D1 molecule is encapsulated. For example,the tubular heat exchanger can have a temperature of about 15° C. toabout 25° C. In one aspect, the method does not employ a gelation bathand thereby avoids the losses associated with such a bath, for examplefrom washing and collecting the gelled beads.

FIG. 2A is a schematic diagram of a non-limiting example of a suitableapparatus for performing this method, FIG. 2B contains photographscomparing the dense powder structure achieved by this method to thehighly porous powder structure obtained by conventional spray drying,and FIG. 2C is a photograph of a non-limiting example of a suitableapparatus for performing this method. The heat exchanger can extrude thehydrogel in a desired shape, for example strands of hydrogel as shown inthe photograph of FIG. 2D.

In an embodiment, the extruded hydrogel is immersed in a compositioncomprising calcium chloride before the drying. In another embodiment,the extruded hydrogel is immersed in a composition comprising potassiumchloride before the drying, for example a bath of about 1 mM potassiumchloride. In one aspect, the extruded hydrogel can be dried in a dryingtunnel or fluid bed drier. The method can comprise milling the driedhydrogel to a predetermined size to form the powder.

For example, the strands (or cut pieces thereof) of extruded materialcan be dried in a fluid bed drier with an inlet air temperature of20-25° C. at an air flow of 120 m³/minute for 30 to 60 minutes, then atabout 40° C. at an air flow of about 120 m³/minute for an additional 60to 90 minutes. The resulting dry material can be collected and eitherused “as-is” or milled to further reduce the particle size to a desiredsize.

As another example, the strands (or cut pieces thereof) of extrudedmaterial can be dried in a drying tunnel with an inlet air temperatureof 20-25° C. at an air flow of 20-80 m³/minute for 30 to 60 minutes,then at about 40° C. at an air flow of 20-120 m³/minute for anadditional 60 to 90 minutes. The resulting dry material can be collectedand either used “as-is” or milled to further reduce the particle size toa desired size.

As noted above, the present inventors surprisingly found that optimizedspray drying conditions and also careful selection of gelatin properties(e.g., molecular weight) and wall materials could develop a formulationcapable of being spray dried, with the resulting powder retaining itsmucoadhesive properties. Therefore, yet another aspect of the presentdisclosure is a food grade powder that provides sustained release of ananti-Fel D1 molecule and comprises a spray-dried hydrogel comprising theanti-Fel D1 molecule and 0.5 to 90.0 wt. % of gelatin. The powdercomprising the spray-dried hydrogel has a density of about 0.1 g/cm³ toabout 0.6 g/cm³, about 0.265 g/cm³ to about 0.5 g/cm³, or in one aspect,about 0.4 g/cm³ to about 0.5 g/cm³. The anti-Fel D1 molecule can beencapsulated in the spray-dried hydrogel. In an embodiment, the gelatincan be the only biopolymer capable of gelling below 60° C. in thespray-dried hydrogel. In some aspects, the gelatin can have a bloomvalue of 40 to 300, or 40 to 200, or even about 100. In one aspect, thegelatin can be Type A gelatin (acid-treated).

In one embodiment, the powder can comprise 10.0 to 99.5 wt. % of asource of the anti-Fel D1 molecule (e.g., partially defatted egg yolkpowder from an avian immunized with Fel D1). In one aspect, the powdercan comprise 10.0 to 80.0 wt. % of the gelatin and 20 to 90 wt. % of thesource of the anti-Fel D1 molecule. In another aspect, the powder cancomprise 25.0 to 80.0 wt. % of the gelatin and 20 to 75 wt. % of thesource of the anti-Fel D1 molecule. In still another aspect, the powdercan comprise 25.0 to 60.0 wt. % of the gelatin and 40 to 75 wt. % of thesource of the anti-Fel D1 molecule.

FIG. 3 shows a non-limiting example of a suitable apparatus for makingthis embodiment of the powder. The method for making this embodiment ofthe powder can comprise spray drying a solution comprising an anti-FelD1 molecule and 5.0 to 20.0 wt. % of gelatin having a bloom value of 40to 200 to form a dried hydrogel in which the anti-Fel D1 molecule isencapsulated. In an embodiment, the gelatin can be the only biopolymerthat is capable of gelling below 60° C. in the solution. In anotherembodiment, the gelatin can have a bloom value of 40 to 200, or in oneaspect, about 100.

The solution can comprise 0.5 to 30.0 wt. % of a source of the anti-FelD1 molecule (e.g., partially defatted egg yolk powder from an avianimmunized with Fel D1). In one aspect, the solution can comprise 2.0 to15.0 wt. % of the gelatin and 2.0 to 20.0 wt. % of the source of theanti-Fel D1 molecule; and in one specific aspect, 7.5 to 12.5 wt. % ofthe gelatin and 5 to 20 wt. % of the source of the anti-Fel D1 molecule.

Yet another aspect of the present disclosure is a method of reducingsymptoms of human allergy to a cat. The method comprises orallyadministering to the cat an effective amount of any of the food gradepowders disclosed herein and/or a food grade powder resulting from anyof the methods disclosed herein. The powder can be administered as partof a pet food further comprising at least one component selected fromthe group consisting of protein, fat, carbohydrate, vitamin and mineral.The method can bind the anti-Fel D1 molecule to the Fel D1 in the cat'smouth and thereby prevent the Fel D1 from inducing an allergic reactionin a human susceptible to or suffering from an allergy caused by Fel D1.

EXAMPLES

By way of example and not limitation, the following non-limitingexamples are illustrative of compositions and methods for reducingsymptoms of human allergy to cats in embodiments provided by the presentdisclosure.

Example 1

A first study was performed to understand the impact of different dryingapproaches on the release of anti-Fel D1 IgY from hydrogels in order tohelp guide the development of manufacturing approaches for the deliverysystem. Two different hydrogel formulations; i) a simple gelatin-onlyformulation and ii) a gelatin/kappa-carrageenan co-gel formulation werecreated using two approaches; i) direct spray drying and ii) beadletformation followed by drying. The central hypothesis was that gelationof the hydrogel before drying would result in a better capsule structurethat is more able to slow the rate of anti-Fel D1 IgY release from thedried hydrogel.

This study found that the type of drying used to create hydrogel powderscan have a major impact on the rate of anti-Fel D1IgY release fromhydrogel microcapsules. It was found that gelling the hydrogelformulation before drying resulted in microcapsules with thicker anddenser wall structures. The resulting “gel→dried” microcapsules had ananti-Fel D1IgY release rate 2.75 times slower than spray driedmicrocapsules. No difference in anti-Fel D1 IgY release rate wasobserved between simple gelatin-only microcapsules and complexgelatin/kappa-carrageenan co-gels. In wet hydrogels ofgelatin/kappa-carrageenan co-gels, the release of anti-Fel D1 IgY fromthe co-gels was ten times slower than from gelatin-only gels.

Previous work within encapsulation assessed several differenttechnologies (i.e., hydrogels, self-assembled structures, liposomes, andwater-in-oil emulsions) to enhance the residence time of anti-Fel D1 IgYin cats' mouths. Overall, type A gelatin biopolymer hydrogels were foundto be a leading technology because they had high encapsulation efficacyand inherent mucoadhesion.

As shown in FIG. 4, a limitation of simple gelatin (10 wt. %) hydrogelsis that they rapidly release all the encapsulated IgY when exposed tooral conditions (37° C. and simulated saliva). However, combining thegelatin hydrogel with a second hydrogel (e.g. agar, carrageenan) wasfound to enable the wet gelatin/kappa-carrageenan hydrogels to releaseanti-Fel D1 IgY over two hours. Surprisingly, all the co-gels had muchslower anti-Fel D1 IgY release kinetics compared to simple hydrogels ofgelatin alone or kappa-carrageenan alone, highlighting the uniquesynergy of combining the two biopolymers.

These hydrogels have high Aw (>0.6) which presents significant risks tothe stability of anti-Fel D1 IgY from microbial spoilage, proteinoxidation/denaturation and enzymatic degradation. Therefore thesehydrogel structures must be dried without losing their ability to have asustained release of anti-Fel D1 IgY over 2 to 4 hours. Hydrogelmatrices can be dried using a number of approaches, each having animpact on the final powder structure and hence IgY release kinetics. Themost conventional approach is spray drying, where the parent solution isdirectly dried in an atomized air stream without the solution passingthrough a gel state. A second approach is “gel→drying” where wet gelparticles of the parent solution (e.g. by extrusion/dripping orprilling) are created before the drying step. The advantage of“gel→drying” approaches is that the structure of the hydrogel is morelikely to be retained.

The effect of different drying techniques on anti-Fel D1 IgY releasefrom hydrogels was first examined using simple hydrogels composed ofgelatin. The parent solution consisted of 10 wt. % gelatin (type A bloom280) and 2 wt. % egg yolk extract. As shown in FIGS. 5A-5D, differentstructures were obtained from this solution depending on whether thesolution dried directly without gelling (FIG. 5B) or gelled beforedrying (FIG. 5D).

As shown in FIGS. 6A and 6B, when the solution was dried directlywithout gelling, the resulting power particles were small, very porousand even hollow (FIG. 6A). Such a powder structure is common for spraydried powders made from solutions of low total solids (12 wt. %). Whenthe gelatin solution was gelled before drying, via dripping, theresulting powder particles were “shrunken” but had a thick, very densepowder wall structure (FIG. 6B).

It was apparent that gelling the hydrogel before drying led to amarkedly different structure than direct drying via spray drying. Inorder to gain insight into how this structure formed, the microstructureof the hydrogel was investigated using cryo-SEM with ice sublimation.FIG. 7A shows an outside view of a gel beadlet, and the only obviouselement of its structure is that it is a sphere with a rough surface. Across sectional analysis of the internal structure of the hydrogel canbe achieved via freeze fracture SEM (FIGS. 7B and 7C). Both FIGS. 7B and7C show that the gelatin hydrogel forms a mesh-like structure with aconcentration of gelatin within the walls of each cell and large voidsin the middle of each cell. Ice sublimation to remove the water in thestructure shows the complexity of the cellular structure of the gelatinhydrogel, where there is a hierarchy of cellular structures that form adense honeycomb matrix.

At initial viewing, one might think that the size of these cells (on themicrometer scale) is too large to limit the transport of IgY. However,examination of the fine structure of the wall of the gelatin gelsreveals a very thick dense network (right panel of FIG. 7B). Upondrying, this cellular structure collapses upon itself to form the verydense walls of the powder particles.

Release of anti-Fel D1 IgY from these different particles was assessedby dispersing the powder in saline at 37° C. to mimic oral conditions.FIG. 8 presents the release of anti-Fel D1 IgY from different hydrogelstructures: (I) wet gel pieces, (II) spray dried particles, (III) wethydrogel beadlets and (IV) dry hydrogel beadlets.

The release of anti-Fel D1 IgY from the control sample, a gelled pieceof the 10 wt. % gelatin, is shown in FIG. 8. Initially there was almostno anti-Fel D1 IgY when the gel piece was immersed in cold saline,showing that it had high IgY encapsulation efficiency (>98%). Uponimmersion in saline at 37° C., there is a rapid increase in the amountof anti-Fel D1 IgY released, reaching 80% after 5 minutes and 100% after15 minutes. Such rapid release is to be expected because 10 wt. %gelatin gels melt at temperatures above 25-30° C.

Likewise, the small wet gelatin beadlets rapidly released anti-Fel D1IgY upon immersion in 37° C. saline, releasing 100% of the IgY over 15minutes, again due to the melting of the hydrogel (FIG. 8). However,when the wet beadlets were dried, the release of anti-Fel D1 IgY slowedconsiderably: after 15 minutes, only 33.5% of IgY was released; at 30minutes, 75% of IgY was released; and at 60 minutes, 100% of the IgY hadbeen released (FIG. 8).

In contrast, directly drying gelatin solutions using spray drying didnot lead to a retardation of IgY release. When spray dried gelatinpowders were immersed in 37° C. saline, 50% of IgY was released after 5minutes and 100% was released after 15 minutes (FIG. 8).

There is a striking difference in the rate of IgY release from spraydried powders compared to beadlets which were dried after gelling. Thisdifference in IgY release is likely due to the difference in powder wallstructure that is created by the two techniques. Spray drying led tocapsules with a very thin wall structure, which would have a relativelyhigh amount of surface area exposed to the saline. In contrast, dryinggelatin solutions after gelation created powder particles which had athick dense wall structure with a much lower relative amount of surfacearea exposed to the saline. It is likely that the lower surface area tovolume ratios and the thicker/denser powder wall structure lead to muchslower dissolution of the powder particles, resulting in much sloweranti-Fel D1 IgY release.

The effect of different drying techniques on anti-Fel D1IgY release fromhydrogels was also examined using complex hydrogel co-gels composed ofgelatin and carrageenan. The parent solution consisted of 10 wt. %gelatin (type A bloom 280), 1 wt. % kappa-carrageenan and 2 wt. % eggyolk extract. The effect of drying co-gel systems was assessed using (i)spray drying and (ii) dripping then fluidized bed drying.

FIG. 9A shows the appearance of a gelatin/kappa-carrageenan system afterit has been spray dried. The spray dried gelatin/kappa-carrageenanpowder consisted of hollow small particles with thin walls. In addition,aggregates were present in the structures which were found to becollections of small individual powder particles bound together by wiresof the solution (FIG. 9B). Such a structure arises due to the highvisco-elasticity of the parent 10 wt. % gelatin, 1 wt. %kappa-carrageenan solution. Highly visco-elastic solutions are difficultto atomize because the fluid bridge between two separating dropletsretards complete droplet break-up resulting in the formation of strands.These strands can form fibers or aggregates by interacting with eachother or the droplets in the spray tower. This was particularly the casewhen a rotating disc was used for atomization of the solution.

FIGS. 10A and 10B respectively show the appearance of wetgelatin/kappa-carrageenan co-beadlets and dry gelatin/kappa-carrageenanco-gel beadlets both produced by dripping the parent solution into 100mM KCl. The process of gelling the beadlets then drying them createspowder particles that have a thick, very dense powder wall structure(FIG. 10C). Most powder particles had numerous air pockets within them,presumably a result of the loss of volume caused by the removal of thewater. Sublimation experiments (not shown) similar to the gelatin-onlysystem indicated that the co-gels had a similar wet beadlet structurecompared to gelatin only systems.

FIG. 11A presents the release profiles of anti-FelD1 IgY from 10 wt. %gelatin/1 wt. % kappa-carrageenan co-gel spray dried powders. Thegelatin/carrageenan co-gel spray dried powder releases 10% of IgY whenit is placed in cold saline, indicating a high IgY encapsulationefficiency (˜90%). However, when the co-gel powder was placed in 37° C.saline release of IgY was rapid, 30% of IgY was released after 5minutes, 90% after 15 minutes and 100% after 30 minutes. This releaserate is comparable to gelatin-only capsules and indicates that thepresence of kappa-carrageenan does not have an impact on IgY release.

FIG. 11A also presents the release profiles of IgY from 10 wt. %gelatin/1 wt. % kappa-carrageenan co-gel beadlets in wet and dry format.Both the wet and dry beadlets exhibit high IgY encapsulation efficiency,releasing <2 wt % of IgY when the beadlets are dispersed in cold saline(4° C.). When the wet beadlets are immersed in 37° C. saline, IgYrelease is rapid: approximately 50% of IgY is released after 5 minutes,and 100% is released after 15 minutes. The dried co-gel beadlets exhibitslower IgY release compared to the wet co-gel beadlets. When the driedco-gel beadlets are placed in 37° C. saline, IgY release is slow: after5 minutes 17% of IgY is released, rising to 40% after 15 minutes and 67%after 30 minutes, total IgY release occurs around 60 minutes.

Gelling the co-gel solution prior to drying results in a considerableslowing of IgY release rate (approx. 2.75 times). However, the IgYrelease rate of the dried co-gel beadlet is comparable to that of thedried gelatin beadlet (FIG. 11B). This indicates that the presence ofkappa-carrageenan does not lead to an additional slowing in IgY release.This result is in contrast to earlier work with wet gels whichdemonstrated that gelatin/carrageenan co-gels had a 10 times slower IgYrelease rate compared to gelatin-only hydrogels (FIG. 11B). The lack ofa slower IgY release rate for dried co-gels compared to dried gelatingel beadlets (both in the dry and wet states) indicates that thecarrageenan gel did not form properly. These beadlets were formed bydripping the co-gel solution into a 100 mM KCl solution because theviscosity of the solution was too high to drip. The original co-gelpieces were created by having the KCl in the co-gel solution prior togelation. It is apparent from the previous experiments that removal ofKCl from the co-gel solution could be a key reason why IgY release wasnot slowed down by the presence of the carrageenan in the co-gel.

In summary, the type of drying used to create hydrogel powders can havea major impact on the rate of IgY release from hydrogel microcapsules.Gelling the hydrogel formulation before drying was found to result inmicrocapsules with thicker and denser wall structures. The resulting“gel→dried” microcapsules had an IgY release rate 2.75 times slower thanspray dried microcapsules. No difference in IgY release rate wasobserved between simple gelatin only microcapsules and complexgelatin/K-carrageenan co-gels. In wet hydrogels,gelatin/kappa-carrageenan co-gels provided release of IgY that was 10times slower than from gelatin only gels.

Example 2

A second experiment was performed to investigate the dripping methoddisclosed herein that employs a gelation bath and the extrusion methoddisclosed herein that does not employ a gelation bath. An anti-Fel D1IgY was utilized in a gelatin-carrageenan co-polymer gel.

An oral delivery vehicle should have high content of the activemolecule. Typical losses of the active molecule occur due to lowencapsulation efficacy and denaturation of the IgY duringprocessing/manufacture. The results of the dripping method and theextrusion method with respect to denaturation and encapsulationefficiency are shown in FIG. 12. Regarding denaturation of the anti-FelD1 IgY, 20 to 60% of the IgY was lost during the manufacturing in thedripping method, and 0 to 15% of the IgY was lost during themanufacturing in the extrusion method. Regarding encapsulation of theanti-Fel D1 IgY, about 20% of the IgY was lost in the bath during thegelation step of the dripping method, and less than 1% of the IgY waslost during the extrusion method.

An oral delivery vehicle should also have controlled release of theactive molecule; release of the anti-Fel D1 IgY in the cat's mouthoccurs via dissolution. Accordingly, release of the anti-Fel D1 IgY wasinvestigated in simulated saliva at 37° C. As shown in FIGS. 13A and13B, the anti-Fel D1 IgY in egg powder had a fast release: 100% releasedin less than 5 minutes (control 1), and the anti-Fel D1 IgY encapsulatedin gelatin alone also had a fast release: 90% in less than 5 minutes(control 2). The gelatin-carrageenan co-polymer gel allowed the time of100% release to be tuned from 30 to 90 minutes when the dripping methodwas used and allowed the time of 100% release to be tuned from 30 to 60minutes when the extrusion method was used.

An oral delivery vehicle should also have oral adhesion becausesustained release of the anti-Fel D1 IgY in the cat's mouth requiresthat the IgY stays in the mouth for some time. Mucoadhesive particlesare thus advantageous because they can adhere to the salivary film. Theoral adhesion of the particles from the extrusion method were comparedto free IgY by investigating the interaction with human salivary film atabout 37° C., specifically by measuring adsorption onto a salivarypellicle at 36.8° C. The results are shown in FIG. 14. Free IgY had nointeraction with salivary film, but the particles from co-gel extrusionhad a moderate increase in the interaction with the salivary film, cleardeposition of particles on film (A mass period D).

Example 3

To further investigate using encapsulation to improve the oral residencetime of an anti-Fel D1 IgY, a third experiment was performed. FIG. 15 isa graph showing the concentration of the anti-Fel D1 IgY in cats' salivaafter they were fed 1 g or 2 g of egg yolk containing anti-Fel D1 IgYwith wet or dry food. Less than 5% of the anti-Fel D1 IgY was detectedin the cat's mouth, the remainder was quickly swallowed.

The oral adhesion of the particles from the controlled spray-dryingmethod were compared to free IgY by investigating the interaction withhuman salivary film at about 37° C., specifically by measuringadsorption onto a salivary pellicle at 36.8° C. The results are shown inFIG. 16. Free IgY had no interaction with salivary film, but theparticles from the controlled spray-drying method had a considerableincrease in the interaction with the salivary film, clear deposition ofparticles on film (Δ mass period D).

Example 4

A fourth experiment was performed in which the spray-dried gelatin/IgYmixture was further analyzed. Without being bound by theory, the presentinventors believe that the mucoadhesion of the spray-dried gelatin/IgYmixture relies on a porous powder structure that allows some initialhydration, which creates mucoadhesion interactions through a combinationof i) electrostatic interactions (positive charge on gelatin) andhydration/competition for water (i.e., the particles are sticky). Theglassy co-gel of gelatin-carrageenan, while still effective, may havelower oral adhesion due to much slower hydration.

If a particle can be made to stick to the oral surface, oral residencetime can be further enhanced by controlling the speed of dissolution.The powders of the dripping and extrusion methods disclosed hereincontrol dissolution by powder porosity and controlled dis-assembly ofcarrageenan gels. The spray-dried gelatin/IgY mixture uses porousgelatin powders which have relatively quick in vitro dissolution, themain focus is maximizing oral adhesion. However, informal feedback fromclinical trials suggests that in vivo dissolution is slower thanobserved in vitro and may be beneficial.

It is well known that creation of a powder from gelatin can bedifficult. The main technical challenge is that the viscoelasticproperties of gelatin solutions limit droplet break-up duringatomization in the drier. If the solution has too high viscosity, or ifthe atomization is too rapid the solution creates filament structuresrather than powder particles. This results in a “fluffy” powder whichhas a low density. A low powder density creates problems during powderhandling, powder stability or when trying to apply it to a product as acoating.

As shown in FIG. 17, to overcome the problems of fluffy gelatin powderswith low density, the present inventors found three factors that canimprove density: (1) gelatin concentration—gelatin concentrations above16% create highly filamentous powders and hence cannot be spray dried(Table 1—TJW 040 series—comparison ii); (2) gelatin molecular weight(bloom strength)—switching to a lower molecular weight gelatin (bloom100 rather than 280) decreases filament formation increasing powderdensity (Table 1—comparison i); and (3) combining with otherproteins/molecules—increasing the amount of active ingredient/excipientdecreases filament formation increasing powder density (Table 1—TJW 040series comparison ii).

Using these strategies the powder density could be increase from anunusable powder of 0.1-0.15 g/cm³ for the TJW 020A sample to TJW 050 Aand 106 powders whose density (0.265-0.3 g/cm³) approaches that ofcommercial whey protein isolate (WPI) dairy powder (0.43 g/cm³).

Example 5

A fifth experiment was performed that assessed the impact ofencapsulation on the oral retention of IgY. Twelve human subjects weregiven two powders [i) IgY control—defatted egg yolk powder and ii)defatted egg yolk powder encapsulated in gelatin (type a, 100 bloom)]using a randomized cross over study design (FIG. 18), and asked to chewthe powder for 30 seconds and then swallow. The composition and dose ofthe two test samples are given FIG. 19. Oral retention of IgY wasassessed by measuring IgY concentration in saliva at baseline and atvarious time points as per clinical trial scheme outlined in FIG. 18.Measures of oral retention were; maximum concentration (C_(max)) andarea under the curve (AUC).

FIG. 20 (Sample i) presents the average change in IgY concentration(ng·ml⁻¹) in participants saliva over time for the control IgY and IgYencapsulated in spray dried gelatin. After consumption of the controlIgY, IgY concentration peaked at 206645±71911 ng·ml⁻¹ at 5 minutes(first time point). Salary IgY concentration then underwent anexponential decrease to 21502±6095 ng·ml⁻¹ at 15 minutes, 3224±905ng·ml⁻¹ at 30 minutes, 1329±480 ng·ml⁻¹ at 60 minutes, plateauing at 200to 300 μg·mL-1 two hours after consumption of the control IgY (defattedegg yolk powder). The average total amount of IgY detected in the humanmouth was between 1.95 mg (single integration) and 3 to 3.5 mg (doubleexponential pk integration). The average amount of IgY detected in thehuman subjects mouth was between 1.6 and 2.8% of the original amountadministered (˜123 mg).

FIG. 20 (Sample ii) also presents the effect of encapsulation on humansalivary IgY concentration. The spray dried encapsulated IgY had aC_(max) of 871,000 ng·mL⁻¹ at 5 minutes which was 4.2 times larger thanthe control IgY (p<0.001). Salivary IgY concentration then underwent anexponential decrease to 81,500 ng·mL⁻¹ at 15 minutes, 10,200 ng·mL⁻¹ at30 minutes, 980 ng·mL⁻¹ at 60 minutes plateauing at 400 to 500 ng·mL⁻¹two hours after consumption of the control IgY (defatted egg yolkpowder). The salivary concentration of IgY after consumption of thespray dried encapsulated IgY was 4 times larger than the control IgY forthe first 30 minutes (p<0.05). Whilst salivary IgY concentration of thespray dried encapsulated IgY and the control IgY were the same 60minutes after ingestion, the spray dried encapsulated salivary IgYconcentration was double the control at 90 and 120 minutes (p<0.15). Thetotal amount of IgY detected for the spray dried encapsulated IgY was7.8 (trapezoid integration) to 13.2 (double exponential pk integration)mg of IgY, which equates to 6.4 to 10.7% of the administered dose. Theseresults highlight that encapsulation of IgY in mucoadhesive gelatinusing spray drying increases the efficacy (both C_(max) and AUC) of IgYby a factor of 4 (p<0.001).

It should be understood that various changes and modifications to thepresently embodiments described herein will be apparent to those skilledin the art. Such changes and modifications can be made without departingfrom the spirit and scope of the present subject matter and withoutdiminishing its intended advantages. It is therefore intended that suchchanges and modifications be covered by the appended claims.

The invention is claimed as follows:
 1. A food grade powder providingsustained release of an anti-Fel D1 molecule, the food grade powdercomprising a dried hydrogel and the anti-Fel D1 molecule; wherein thedried hydrogel comprises gelatin, collagen peptides, or gelatin andcollagen peptides; and carrageenan; wherein the anti-Fel D1 molecule isencapsulated in the dried hydrogel.
 2. The food grade powder of claim 1,wherein the gelatin or the gelatin and collagen peptides are from Type Aporcine gelatin having a positive charge at a pH below 7.0.
 3. The foodgrade powder of claim 1, wherein the carrageenan is selected from thegroup consisting of (i) kappa-carrageenan, (ii) iota-carrageenan, (iii)a physical mixture of kappa-carrageenan and iota-carrageenan and (iv) aco-polymer of kappa-carrageenan and iota-carrageenan.
 4. The food gradepowder of claim 1, wherein the gelatin and the carrageenan are the onlybiopolymers in the dried hydrogel.
 5. The food grade powder of claim 1,wherein the dried hydrogel is a spray-dried hydrogel comprising theanti-Fel D1 molecule and 0.5 to 90 wt. % of gelatin, and the powder hasa density of about 0.265 to about 0.3 g/cm³.
 6. The food grade powder ofclaim 5, wherein the gelatin and collagen peptides have a molecularweight between 2,000 and 1 million Daltons and a bloom value of 40 to300.
 7. The food grade powder of claim 5, comprising 25.0 to 60.0 wt. %of the gelatin and 40 to 75 wt. % of a source of the anti-Fel D1molecule.
 8. A method of making a food grade powder providing sustainedrelease of an anti-Fel D1 molecule, the method comprising: heating asolution comprising gelatin, carrageenan and the anti-Fel D1 molecule;forming droplets of the heated solution; gelling the droplets bysubjecting the droplets to a gelling bath comprising salt or oil to formgelled beads in which the anti-Fel D1 molecule is encapsulated; anddrying the gelled beads to form the food grade powder.
 9. The method ofclaim 8, wherein the gelling bath comprises about 100 mM of potassiumchloride, about 100 mM calcium chloride, or both.
 10. The method ofclaim 8, wherein the drying comprises subjecting the beads to fluid beddrying.
 11. The method of claim 8, wherein the gelatin and thecarrageenan are the only biopolymers capable of gelling below 60° C. inthe dried hydrogel.
 12. A method of making a food grade powder providingsustained release of an anti-Fel D1 molecule, the method comprising:heating a solution comprising gelatin, carrageenan, and the anti-Fel D1molecule; extruding the heated solution to cool and shape the heatedsolution and form an extruded hydrogel in which the anti-Fel D1 moleculeis encapsulated; and drying the extruded hydrogel.
 13. The method ofclaim 12, wherein the extruding is performed by a tubular heat exchangerhaving a temperature of about 0.1° C. to about 35° C.
 14. The method ofclaim 12, further comprising immersing the extruded hydrogel in acomposition comprising calcium, magnesium potassium, rubidium, or cesiumbefore the drying.
 15. The method of claim 12, wherein the dryingcomprises subjecting the extruded hydrogel to a drying tunnel, oven, orfluid bed drier.
 16. A method of making a food grade powder providingsustained release of an anti-Fel D1 molecule, the method comprises spraydrying a solution comprising the anti-Fel D1 molecule and 5.0 to 20.0wt. % of gelatin and collagen peptides having a molecular weight between2,000 and 1 million Daltons and or a bloom value of 40 to 300 to form adried hydrogel in which the anti-Fel D1 molecule is encapsulated. 17.The method of claim 16, wherein the gelatin and collagen peptides arethe only biopolymers capable of gelling below 60° C. in the driedhydrogel.
 18. The method of claim 16, wherein the solution comprises 7.5to 12.5 wt. % of the gelatin and 5 to 20 wt. % of a source of theanti-Fel D1 molecule.
 19. A method of reducing symptoms of human allergyto a cat, the method comprising orally administering to the cat aneffective amount of the food grade powder of claim
 1. 20. The method ofclaim 19, wherein the composition is administered as part of a pet foodfurther comprising at least one component selected from the groupconsisting of protein, fat, carbohydrate, vitamin and mineral.